Cancer is a disease that is caused by the non-limiting growth of cells which have lost the control of the cell cycle. Since cancer cells generally grow faster than normal cells, it is considered that cancer can be treated by the control of cell division and DNA replication. In fact, gemcitabine having a DNA replication inhibitory effect is widely used in the treatment of non-small cell lung cancer, pancreatic cancer, bile duct cancer, urinary bladder cancer, breast cancer, or ovary cancer, etc.
Various proteins are involved in the process of the cell cycle. In general, the biological functions of proteins are regulated by various mechanisms of post-translational modifications. Specifically, methylation, acetylation, glycosylation, phosphorylation, and the like are involved in the functional or structural modifications of proteins. Among these post-translational modifications, the phosphorylation is an important mechanism related to the regulation of many functions such as intracellular signal transduction, cell cycle, and cell death. For example, ⅓ or more of the intracellular proteins of mammalian cells are deemed to be phosphorylated.
Protein kinases are enzymes that catalyze the reaction of bonding a phosphoric acid group to a particular amino acid residue in their substrate proteins. This action of the protein kinases phosphorylates a protein at a particular phosphorylation site. The protein kinases are classified as follows on the basis of the types of amino acids at sites to be phosphorylated:
serine-threonine kinase (which phosphorylates a Ser/S or Thr/T residue) and
tyrosine kinase (which phosphorylates Tyr/Y)
Cdc7, a serine-threonine kinase, is an essential protein kinase that participates in the initiation of DNA replication in the cell cycle. Cdc7 forms a complex with a cofactor, such as Dbf4 (ASK), which activates its phosphorylating effect, and phosphorylates a substrate MCM (minichromosome maintenance) protein. This phosphorylation appears to allow Cdc45 and DNA polymerase to assemble on DNA so that an MCM complex is formed to start DNA replication (see Non-Patent Document 1).
In recent years, Cdc7 has received attention as a target of anticancer agents and has been actively studied. For example, Cdc7 has been found to be overexpressed not only in general human tumor-derived cell lines but in cancer cells, such as breast cancer, colon cancer, and lung cancer cells, collected from organisms (see Non-Patent Document 2). Particularly, it has recently been shown that Cdc7 is overexpressed in triple negative (ER-/PR-/Her2-) breast cancer cells having p53 mutation (see Non-Patent Document 3). Cdc7 is thus expected to serve as a promising target for triple negative-type breast cancer, which has been regarded as being difficult to treat. In fact, a Cdc7 expression suppression experiment using an RNA interference technique has demonstrated that the inhibition of Cdc7 expression induces the arrest of the cell cycle of normal cells. More importantly, the inhibition of Cdc7 based on the RNA interference technique inhibited the growth of human tumor cells such as HeLa and HCT116, but was less effective for inhibiting normal cells (normal human skin fibroblasts) (see Non-Patent Document 4).
The deletion of DNA replication factors often brings about cell death to cancer cells. Since Cdc7 is also an essential protein kinase involved in the initiation of DNA replication in the cell cycle, its deletion induces the death of cancer cells, regardless of the status of the p53 gene that controls the suppression of the cell growth cycle involving intracellular DNA repair, cell growth arrest, apoptosis, etc. Masai et al. have utilized a fluorescent probe of recently developed Fucci (fluorescent ubiquitination-based cell cycle indicator) to study cell death induced by the deletion of Cdc7 while observing the progression of the cell cycle at real time (see Non-Patent Document 5). They have showed that the inhibition of Cdc7 induces clear cell cycle response in both of p53-positive and p53-negative cells. Particularly, in the p53-negative cells, the progression of the cell cycle is temporarily arrested in the G2-phase prior to the M-phase so that cyclin B1 and other mitotic control gene products accumulate. Subsequently, the G2-phase proceeds to the abnormal M-phase, and cell death occurs after the mitosis. On the other hand, in the p53-positive cancer cells, cyclin B1 does not accumulate, whereas most of the cancer cells are reportedly killed by transition to the abnormal S-phase after the deletion of Cdc7 (see Non-Patent Document 5).
Thus, inhibitors that selectively inhibit Cdc7 are expected to exhibit an effective therapeutic effect on various cancers. Various compounds having a Cdc7 inhibitory effect have been reported so far (see Patent Document 1). Particularly, furanone derivatives having a Cdc7 inhibitory effect have been reported (see Patent Document 2).
Wee1 is a nuclear protein kinase that belongs to protein kinases of the serine-threonine family and plays a role as a regulator important for the progression of the cell cycle together with a dephosphorylating enzyme Cdc25.
The Wee1 kinase suppresses the activity of Cdk1 kinase through the phosphorylation of its Tyr14 and Tyr15, whereas Cdc25 dephosphorylates and activates these tyrosine residues. Wee1 or Cdc25 undergoes activation or suppression by the G2/M checkpoint mechanisms. Specifically, the transition from the G2-phase to the M-phase is controlled by the balance between the functions of these enzymes Wee1 and Cdc25 located downstream of the checkpoint.
From the aforementioned functions of Wee1, it is considered that the inhibition of Wee1 can cause the transition of abnormal or defective cells to the M-phase, leading to immature mitosis or cell death. The application of Wee1 inhibitors to anticancer treatment has therefore been deliberated. In fact, a Wee1 inhibitor MK-1775 has been reported to have an anticancer effect (see Non-Patent Document 6). Its anticancer effect or selectivity for cancer cells, however, is less than satisfactory.
On the other hand, a Wee1 inhibitor MK-1775 developed by Merck KGaA has been reported to enhance the cytotoxic effect of a DNA-damaging agent such as gemcitabine on human sarcoma (Non-Patent Document 7). Unlike the DNA-damaging agent such as gemcitabine, Cdc7 inhibitors do not directly cause DNA damage. Hence, no conventional technique has disclosed the combination of a Cdc7 inhibitor and a Wee1 inhibitor or reported its anticancer effect on cancer cells or cancer cell selectivity.